The present application is the national stage under 35 U.S.C. 371 of international application PCT/IL99/00605, filed Nov. 10, 1999 which designated the United States, and which international application was published under PCT Article 21(2) in the English language. The entire contents of said PCT/IL99/00605 are hereby incorporated by reference.
The present invention relates to red colored covalent conjugates of 4xe2x80x2-hydroxyazobenzene-2-carboxylic acid derivatives (hereinafter HABA) and an avidin-type molecule, these conjugates referred herein as xe2x80x9cHABAylated avidinsxe2x80x9d, and their application in the avidin-biotin technology.
Avidin and streptavidin are tetrameric proteins that, due to their strong interaction with biotin, have become widely useful as extremely versatile, general mediators in a broad variety of bioanalytical applications, including chromatography, cytochemistry, cell cytometry, diagnostics, immunoassays and biosensing, gene probes and drug delivery (Wilchek and Bayer, 1990). The reason for their popularity is based on the basic principle of the avidin-biotin technology, namely, that the interaction between avidin and biotin is not affected when biotin is covalently bound to macromolecules or to insoluble carriers (matrices). The biotin moiety can be recognized by the native avidin molecule or derivatized avidin, which contains desired reporter groups.
Avidin (from egg-white) and streptavidin (from Streptomyces avidinii) are two related proteins that bind biotin with similar dissociation constants of about 10xe2x88x9215M. In addition to the binding of biotin, many of their physical properties are quite similar. Both, for example, are constructed of four non-covalently attached identical subunits, each of which bears a single biotin-binding site. The subunit Mr values are also very similar. Moreover, several short stretches in the sequences of the two proteins are preserved, particularly two Trp-Lys stretches that occur at approximately similar positions.
Despite these similarities, several differences exist between the two proteins. Avidin is a disulphide-bridged glycoprotein containing two methionine residues, whereas streptavidin is not glycosylated and is devoid of sulphur-containing amino acid side chains. Another significant difference is in the tyrosine content. Avidin has only one tyrosine residue (Tyr-33), whereas streptavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosine residue of avidin is located in a region which contains a sequence identical with that of one of the streptavidin tyrosine residues (Tyr-43 in the stretch Thr-Gly-Thr-Tyr).
Each avidin monomer binds one molecule of biotin. The unique feature of this binding, of course, is the strength and specificity of formation of the avidin-biotin complex. The resultant affinity constant, estimated at 1.6xc3x971015 Mxe2x88x921 for avidin and 2.5xc3x971013 Mxe2x88x921 for streptavidin, is the highest known for a protein and an organic ligand. It is so strong that biotin cannot be released from the binding site, even when subjected to a variety of drastic conditions such as high concentrations of denaturing agents at room temperature, e.g., 6 M guanidinium hydrochloride, 3 M guanidinium thiocyanate, 8 M urea, 10% xcex2-mercaptoethanol or 10% sodium dodecyl sulfate. Under combined treatment with guanidinium hydrochloride at low pH (1.5) or upon heating ( greater than 70xc2x0 C.) in the presence of denaturing agents or detergents, the protein is denatured, and biotin is dislodged from the disrupted binding site.
Avidin recognizes biotin mainly at the ureido (urea-like) ring of the molecule. The interaction between the binding site of avidin with the sulfur-containing ring of the valeric acid side chain of the vitamin is of much lower strength. The relatively weak interaction between the carboxy-containing side chain of biotin and avidin means that the former can be modified chemically and attached to a wide variety of biologically active material; the biotin moiety of the resultant derivative or conjugate is still available for interaction with avidin. In turn, the avidin can be derivatized with many other molecules, notably xe2x80x9cprobesxe2x80x9d or reporter groups of different types.
This is the crux of avidin-biotin technology (Wilchek and Bayer, 1990). Thus, a biologically active target molecule in an experimental system can be xe2x80x9clabeledxe2x80x9d with its biotinylated counterpart (a binder), and the product can then be subjected to interaction with avidin, either derivatized or conjugated with an appropriate probe.
The use of the egg-white avidin in the avidin-biotin technology is sometimes restricted due to the high basicity (pI 10.5) and presence of sugar moieties on the avidin molecule, which may lead to nonspecific or otherwise undesired reactions. In recent years, the bacterial protein, streptavidin, has largely replaced egg-white avidin for most applications in avidin-biotin technology. However, the problems with streptavidin (high cost and biotin-independent cell binding) have prompted renewed interest in egg-white avidin as the standard for avidin-biotin technology. For this purpose, modified avidins exhibiting improved molecular characteristics both over the native protein (and previous derivatives thereof) as well as over streptavidin, have been prepared, such as N-acyl avidins, e.g., N-formyl, N-acetyl and N-succinyl avidins. These derivatives of avidin reduce the charge of the protein, but they are all prepared via covalent attachment to the available lysines of avidin, and the consequent blocking of the free amino groups hinders subsequent preparation of other types of conjugates (notably protein-protein conjugates such as avidin-labeled enzymes) which are often prepared by crosslinking via lysine residues using bifunctional reagents (e.g. glutaraldehyde).
A more useful and effective alternative to lysine modification is the modification via arginines. In this case, the pI of the protein is efficiently reduced and the lysines are still available for subsequent interaction. Two different derivatives of avidin which are modified in this manner are commercially available. One, ExtrAvidin(copyright), can be obtained in various functionally derivatized or conjugated forms from Sigma Chemical Company (St. Louis, Mo.). A second , NeutraLite Avidin(trademark) (a product of Belovo Chemicals, Bastogne, Belgium) is additionally modified and can be purchased in bulk quantities.
Although the reduction of the pI of egg-white avidin solves one of the problems, the presence of the oligosaccharide residue remains a serious source of nonspecific (biotin-independent) interaction which restricts its application. The return of egg-white avidin as the standard for avidin-biotin technology has been contingent upon the removal of its sugars. The possibilities for removing a sugar from a glycoprotein are quite limited; it is possible to do so either chemically or enzymatically. The chemical methods currently available, e.g., using HF or periodate oxidation, are either destructive or inefficient. The well known enzymatic method, which employs N-glycanase, is usually very expensive and not very effective for avidin when conventional methodology is used. Eventually, a viable procedure for deglycosylation was established and the resultant product was subsequently modified chemically via the arginines and is known under the trade mark NeutraLite Avidin(trademark) (Belovo Chemicals).
In spite of all these improvements, one of the main problems in the several applications of the avidin-biotin technology is the lack of an appropriate labeled avidin to permit the follow up of the binding of avidin to biotinylated compounds.
In addition to its interaction with biotin, avidin is known to associate non-covalently also with 4xe2x80x2-hydroxyazobenzene-2-carboxylic acid at the same biotin-binding site of the protein, but with a lower affinity (xcx9c10xe2x88x925-10xe2x88x926 M) (Green, 1965). This non-covalent association is accompanied by a change in color from yellow to red (350 nm to 500 nm), thus allowing determination of avidin and its free binding sites.
Derivatives of 4xe2x80x2-hydroxyazobenzene-2-carboxylic acid and conjugates thereof with oligo and macromolecular carriers (HABAylated molecules) are the subject of copending application of Applicants filed at the same date as the present application.
It has now been found in accordance with the present invention that certain HABA derivatives covalently bound to avidins, preferably at the binding site, form red colored HABAylated avidins (red avidins) that change the red color to yellow upon binding biotin. The displacement of the HABA moiety out of the binding site by biotin is due to a higher affinity of biotin to the red colored avidin.
The present invention thus relates, in one aspect, to covalent conjugates of 4xe2x80x2-hydroxyazobenzene-2-carboxylic acid derivatives (hereinafter HABA) and an avidin-type molecule, these conjugates referred herein as xe2x80x9cHABAylated avidinsxe2x80x9d, of the formula: 
wherein
A is (CH2)n or xe2x80x94CHxe2x95x90CHxe2x80x94, wherein n is an integer from 0-10;
B is (CH2)n wherein n is an integer from 2 to 10;
m is zero or 1; and
Av is the residue of an avidin-type molecule selected from the group comprising native egg-white avidin, recombinant avidin, deglycosylated avidins, bacterial streptavidin, recombinant streptavidin, truncated streptavidin and other derivatives of said avidin-type molecules.
When the HABA moiety is inside the avidin binding pocket, it has the quinone conformation, the conjugate has a red color and xcexmax=504 nm: this is the HABAylated red avidin. When the HABA moiety is expelled from the avidin binding pocket by biotin, it has the azo configuration, the conjugate has a yellow color and xcexmax=356 nm: this is the HABAylated yellow avidin (see Appendix A). In the specification and claims herein, the term xe2x80x9cHABAylated avidinxe2x80x9d comprises both the azo and the quinone conformations.
In the HABAylated avidins according to the invention, A is preferably xe2x80x94CH2xe2x80x94CH2xe2x80x94 or xe2x80x94CHxe2x95x90CHxe2x80x94, and B is preferably (CH2)2, (CH2)5 or (CH2)6.
The HABAylated avidins of the invention are prepared by reaction of an avidin-type molecule with a succinimidyl ester or carbamate of HABA derivatives of formulas I and II, respectively, or a cyclic derivative of formula III: 
wherein A is (CH2)n or xe2x80x94CHxe2x95x90CHxe2x80x94, wherein n is an integer from 0-10; and B is (CH2)n wherein n is an integer from 2 to 10.
The HABA compounds of formulas I and II are the subject of copending PCT application of Applicants filed at the same date as the present PCT application. The cyclic derivatives of formula III are encompassed by the present invention.
The invention further relates to columns containing immobilized HABAylated avidins, attached to a solid support or matrix.
In another aspect, the invention relates to a single-layer protein system comprising:
(i) a protein:
(ii) two ligands I and II which bind with different affinities at the same binding site of said protein, said ligand I being the low affinity ligand and said ligand II being the high affinity ligand: and
(iii) a molecule that recognizes the low affinity ligand I,
wherein in said single-layer protein system the high affinity ligand II is buried within the binding site of the protein (i) and the low affinity ligand I is covalently bound to the protein and associated with the molecule (iii) that recognizes it.
In one embodiment of the single-layer protein system aspect, the molecule (iii) may be labeled with high affinity ligand II.
In another aspect, the invention relates to a multilayer protein system comprising two or more layers of the single-layer protein system. In the last layer of the multilayer protein system, the molecule (iii) is preferably not labeled with high affinity ligand II.
In one preferred embodiment, the protein (i) in the single-layer or multilayer protein systems is an avidin-type molecule selected from the group comprising native egg-white avidin, recombinant avidin, deglycosylated avidins, bacterial streptavidin, recombinant streptavidin, truncated streptavidin and other derivatives of said avidin-type molecules; the low affinity ligand I is HABA (4xe2x80x2-hydroxyazobenzene-2-carboxylic acid) or a HABA derivative, the high affinity ligand II is biotin, and the molecule (iii) that recognizes ligand I is an anti-HABA antibody or a biotinylated anti-HABA antibody.
The anti-HABA antibody used according to the invention may be polyclonal or monoclonal, and can be prepared by immunization of rabbits and mice, respectively, with a conjugate of HABA and an immunogenic protein, such as for example HABA-KLH. The anti-HABA antibodies are the subject of copending application of Applicants filed at the same date as the present application.
In another embodiment, the protein (i) in the single-layer or multilayer protein system is anti-dinitrophenyl (DNP)-antibody; the low affinity ligand I is trinitrobenzene (TNP) or mononitrobenzene (MNP), the high affinity ligand I is DNP and the molecule (iii) that recognizes ligand I is a MNP- or TNP-tagged anti-DNP antibody.
The single-layer or multilayer protein system according to the invention may be formed on a substrate such as gold, silicium, polystyrene. Preferably, the multilayer protein system will comprise 5-6 layers.
In another aspect, it is provided a method for assembling a single-layer protein system according to the invention, which comprises the steps of:
(a) covalently binding said low affinity ligand I to said protein (i), thus obtaining a low affinity ligand I-protein (i) complex in which said ligand I is buried within the binding site of said protein (i) and is thus not available for interaction with other molecules that recognize it;
(b) reacting the high affinity ligand II or a compound containing said high affinity ligand II with the low affinity ligand I-protein (i) complex of step (a) above, whereby low affinity ligand I is expelled from within the binding site to the periphery but remains covalently bound to protein (i) and high affinity ligand II is associated to, and buried within, the binding site of protein (i); and
(c) reacting the low affinity ligand I-protein(i)-high affinity ligand II complex of step (b) with a molecule (iii) that recognizes and binds to low affinity ligand I and can be labeled with high affinity ligand II.
In still another aspect, it is provided a method for assembling a multilayer protein system according to claim 3, which comprises the steps of:
(a) covalently binding said low affinity ligand I to said protein (i), thus obtaining a low affinity ligand I-protein (i) complex in which said ligand I is buried within the binding site of said protein (i) and is thus not available for interaction with other molecules that recognize it;
(b) reacting the high affinity ligand II or a compound containing said high affinity ligand II with the low affinity ligand I-protein (i) complex of step (a) above, whereby low affinity ligand I is expelled from within the binding site to the periphery but remains covalently bound to protein (i) and high affinity ligand II is associated to, and buried within, the binding site of protein (i);
(c) reacting the low affinity ligand I-protein(i)-high affinity ligand II complex of step (b) with a molecule (iii) that recognizes and binds to low affinity ligand I and is labeled with high affinity ligand II; and
(d) reacting the protein system of step (c) with low affinity ligand I-protein (i) complex as in step (b) above, and repeating steps (c) and (d) as desired.